Conformational Selection of a Tryptophan Side Chain Drives the Generalized Increase in Activity of PET Hydrolases through a Ser/Ile Double Mutation.

Abstract

Poly(ethylene terephthalate) (PET) is the most common polyester plastic in the packaging industry and a major source of environmental pollution due to its single use. Several enzymes, termed PET hydrolases, have been found to hydrolyze this polymer at different temperatures, with the enzyme from Ideonella sakaiensis (IsPETase) having optimal catalytic activity at 30-35 °C. Crystal structures of IsPETase have revealed that the side chain of a conserved tryptophan residue within an active site loop (W185) shifts between three conformations to enable substrate binding and product release. This is facilitated by two residues unique to IsPETase, S214 and I218. When these residues are inserted into other PET hydrolases in place of the otherwise strictly conserved histidine and phenylalanine residues found at their respective positions, they enhance activity and decrease T opt. Herein, we combine molecular dynamics and well-tempered metadynamics simulations to investigate dynamic changes of the S214/I218 and H214/F218 variants of IsPETase, as well as three other mesophilic and thermophilic PET hydrolases, at their respective temperature and pH optima. Our simulations show that the S214/I218 insertion both increases the flexibility of active site loop regions harboring key catalytic residues and the conserved tryptophan and expands the conformational plasticity of this tryptophan side chain, enabling the conformational transitions that allow for substrate binding and product release in IsPETase. The observed catalytic enhancement caused by this substitution in other PET hydrolases appears to be due to conformational selection, by capturing the conformational ensemble observed in IsPETase.